Chemical amplification based on fluid partitioning

ABSTRACT

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

More than one reissue application has been filed for the reissue of U.S.Pat. No. 7,041,481, the reissue applications are application Ser. No.16/115,187, filed Aug. 28, 2018, which is a continuation reissueapplication of reissue of application Ser. No. 15/421,141, filed Jan.31, 2017, now U.S. Pat. No. Re. RE47,080, issued Oct. 8, 2018, which isa continuation reissue application of reissue application Ser. No.14/701,392, filed Apr. 30, 2015, now U.S. Pat. No. Re. RE46,322, issuedFeb. 28, 2017, which is a continuation reissue application ofapplication Ser. No. 13/436,693, filed Mar. 30, 2012, now U.S. Pat. No.Re. 45,539, issued Jun. 2, 2015 which is a continuation reissueapplication of application Ser. No. 12/891,733, filed Sep. 27, 2010,issued as U.S. Pat. No. Re. 43,365, which is a continuation reissueapplication of reissue application Ser. No. 12/118,418, filed May 9,2008, issued as U.S. Pat. No. Re. 41,780, which is a reissue applicationof U.S. Pat. No. 7,041,481. The present application is a Reissue ofapplication Ser. No. 10/389,130, filed Mar. 14, 2003, issued as U.S.Pat. No. 7,041,481 on May 9, 2006 and adds new claims relative to U.S.Pat. No. 7,041,481.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.This invention was made with governmentsupport under W-7405-ENG-48 awarded by United States Department ofEnergy. The government has certain rights in the invention.

BACKGROUND

1. Field of Endeavor

The present invention relates to chemical amplification and moreparticularly to chemical amplification based on fluid partitioning.

2. State of Technology

U.S. Pat. No. 4,683,202 issued Jul. 28, 1987; U.S. Pat. No. 4,683,195issued Jul. 28, 1987; and U.S. Pat. No. 4,800,159 issued Jan. 24, 1989to Kary B. Mullis et al provide background information. The patentsdescribe processes for producing any particular nucleic acid sequencefrom a given sequence of DNA or RNA in amounts which are large comparedto the amount initially present. The DNA or RNA may besingle-or-double-stranded, and may be a relatively pure species or acomponent of a mixture of nucleic acids. The process utilizes arepetitive reaction to accomplish the amplification of the desirednucleic acid sequence. The extension product of one primer whenhybridized to the other becomes a template for the production of thedesired specific nucleic acid sequence, and vice versa, and the processis repeated as often as is necessary to produce the desired amount ofthe sequence.

U.S. Pat. No. 6,503,715 for a nucleic acid ligand diagnostic biochipissued Jan. 7, 2003 provides the following background information,“Methods are provided in the instant invention for obtaining diagnosticand prognostic Nucleic acid ligands, attaching said ligands to aBiochip, and detecting binding of target molecules in a Bodily to saidBiochip-bound Nucleic acid ligands.” In one embodiment of the instantinvention, one or more Nucleic acid ligands are chosen that bind tomolecules known to be diagnostic or prognostic of a disease; theseligands are then attached to the Biochip. Particular methods forattaching the Nucleic acid ligands to the Biochip are described below inthe section entitled “Fabrication of the Nucleic Acid Biochip.” TheBiochip may comprise either (i) Nucleic acid ligands selected against asingle target molecule; or more preferably, (ii) Nucleic acid ligandsselected against multiple target molecules.

U.S. Patent Application No. 2002/0197623 for nucleic acid detectionassays published Dec. 26, 2002 provides the following backgroundinformation, “means for the detection and characterization of nucleicacid sequences, as well as variations in nucleic acid sequences . . .methods for forming a nucleic acid cleavage structure on a targetsequence and cleaving the nucleic acid cleavage structure in asite-specific manner. The structure-specific nuclease activity of avariety of enzymes is used to cleave the target-dependent cleavagestructure, thereby indicating the presence of specific nucleic acidsequences or specific variations thereof.”

SUMMARY

Features and advantages of the present invention will become apparentfrom the following description. Applicants are providing thisdescription, which includes drawings and examples of specificembodiments, to give a broad representation of the invention. Variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this descriptionand by practice of the invention. The scope of the invention is notintended to be limited to the particular forms disclosed and theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

The present invention provides an apparatus for nucleic acidamplification of a sample comprising means for partitioning the sampleinto partitioned sections and means for performing PCR on thepartitioned sections of the sample. Another embodiment of the inventionprovides an apparatus for nucleic acid amplification and detection of asample comprising means for partitioning the sample into partitionedsections, means for performing PCR on the partitioned sections of thesample, and means for detection and analysis of the partitioned sectionsof the sample. The present invention also provides a method of nucleicacid amplification of a sample comprising the steps of partitioning thesample into partitioned sections and subjecting the partitioned sectionsof the sample to PCR. Another embodiment of a method of the presentinvention provides a method of nucleic acid amplification and detectionof a sample comprising the steps of partitioning the sample intopartitioned sections, subjecting the partitioned sections of the sampleto PCR, and detecting and analyzing the partitioned sections of thesample.

The invention is susceptible to modifications and alternative forms.Specific embodiments are shown by way of example. It is to be understoodthat the invention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate specific embodiments of theinvention and, together with the general description of the inventiongiven above, and the detailed description of the specific embodiments,serve to explain the principles of the invention.

FIG. 1 is a flow diagram illustrating one embodiment of a systemconstructed in accordance with the present invention.

FIG. 2 is a flow diagram illustrating another embodiment of a systemconstructed in accordance with the present invention.

FIG. 3 is a diagram of another embodiment of a system constructed inaccordance with the present invention.

FIG. 4 is a diagram of another embodiment of a system constructed inaccordance with the present invention.

FIG. 5 is a diagram of another embodiment of a system constructed inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, to the following detailed description,and to incorporated materials; detailed information about the inventionis provided including the description of specific embodiments. Thedetailed description serves to explain the principles of the invention.The invention is susceptible to modifications and alternative forms. Theinvention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

Referring now to the drawings, and in particular to FIG. 1, a flowdiagram of one embodiment of a system constructed in accordance with thepresent invention is illustrated. The system is designated generally bythe reference numeral 100. The system 100 provides a method andapparatus for performing extremely rapid nucleic acid amplification. Theflow diagram illustrating system 100 shows block 101 “partitioning” thesample and block 102 performing “CR” on the sample. The system 100provides an apparatus for nucleic acid amplification of a samplecomprising means for partitioning the sample and means for performingPCR on the sample. The system 100 also provides a method of nucleic acidamplification of a sample comprising the steps of partitioning thesample and subjecting the sample to PCR. The system 100 has applicationwherever current PCR-type systems exist.

In block 101 a chemical reagent and an input sample are “partitioned”into a large number of microdroplets or other forms of fluid partitionsprior to amplification in block 102. The partitioning 101 involvesdispersing the DNA-containing solution. For example the partitioning 101can be accomplished by dispersing the DNA-containing solution in animmiscible carrier liquid. The DNA-containing solution is dispersed inthe immiscible carrier fluid as microdroplets. The DNA-containingsolution can be partitioned in other ways, for example, by beingdispersed as liquid slugs separated by the carrier fluid, as an emulsionwith the carrier fluid, or by using a gelling agent that preventstransfer of DNA between partitioned regions. The DNA-containing solutioncan also be partitioned mechanically by partitioning the fluid intomicro-tubes or capillaries, or into micro-wells.

With the system 100, each partitioned DNA-containing fluid volumecontains the necessary biochemical constituents for selectivelyamplifying a specified portion of a sample DNA via polymerase chainreaction (PCR). The target DNA can be detected by monitoring for thecolorimetric indicator (e.g., flourescence or optical absorption)generated with each DNA template duplicaton sequence.

In block 102 selected portions of each nucleic acid sample are amplifiedusing polymerase chain reaction (PCR), with the product contained ineach partitioned fluid volume. This results in much more concentratedamplification product, since the volume containing the reaction is sosmall.

The polymerase chain reaction (PCR), is a cyclic process whereby a largequantity of identical DNA strands can be produced from one originaltemplate. The procedure was developed in 1985 by Kerry Mullis, who wasawarded the 1993 Nobel prize in chemistry for his work. In PCR, DNA isimmersed in a solution containing the enzyme DNA polymerase, unattachednucleotide bases, and primers, which are short sequences of nucleotidesdesigned to bind with an end of the desired DNA segment. Two primers areused in the process: one primer binds at one end of the desired segmenton one of the two paired DNA strands, and the other primer binds at theopposite end on the other strand. The solution is heated to break thebonds between the strands of the DNA, then when the solution cools, theprimers bind to the separated strands, and DNA polymerase quickly buildsa new strand by joining the free nucleotide bases to the primers in the5′-3′ direction. When this process is repeated, a strand that was formedwith one primer binds to the other primer, resulting in a new strandthat is restricted solely to the desired segment. Thus the region of DNAbetween the primers is selectively replicated. Further repetitions ofthe process can produce a geometric increase in the number of copies,(theoretically 2n if 100% efficient whereby n equals the number ofcycles), in effect billions of copies of a small piece of DNA can bereplicated in several hours.

A PCR reaction is comprised of (a) a double-stranded DNA molecule, whichis the “template” that contains the sequence to be amplified, (b)primer(s), which is a single-stranded DNA molecule that can anneal(bind) to a complimentary DNA sequence in the template DNA; (c) dNTPs,which is a mixture of dATP, dTTP, dGTP, and dCTP which are thenucleotide subunits that will be put together to form new DNA moleculesin the PCR amplification procedure; and (d) Taq DNA polymerase, theenzyme which synthesizes the new DNA molecules using dNTPs.

Current amplification systems are limited in practice to half hour typeamplification and detection windows (−30 cycles, 1 minute/cycle). Thesystem 100 provides faster amplification. This has many applications,for example, in Homeland Defense applications, faster detection methods(a few minutes) can push the deployment of these sensors from “detect totreat” to “detect to protect,” having a serious impact on the number ofcasualties from a massive bioagent release.

The system 100 has significant advantages over typical bulk DNAdetection techniques (even microscale bulk solution approaches),including (1) much faster detection time through a reduction in thetotal number of temperature cycles required, (2) a reduction in the timefor each cycle, and (3) removing interference from competing DNAtemplates. The system 100 achieves a reduction in the total number ofcycles by limiting the dilution of the optically generated signal (e.g.,fluorescence or absorption). The formation of partitioned fluid volumesof the DNA-containing solution effectively isolates the fluid volumeswhich contain the target DNA from the fluid volumes that do not containthe target DNA. Therefore, the dilution of the optical signal is largelyeliminated, allowing much earlier detection. This effect is directlyrelated to the number of fluid partitions formed from the initialsample/reagent pool.

The system 100 achieves a reduction in the total number of cycles thatare needed by limiting the dilution of the optically generated signal(e.g., fluorescence or absorption). The formation of partitioned fluidvolumes of the DNA-containing solution effectively isolates the fluidvolumes which contain the target DNA from the fluid volumes that do notcontain the target DNA. Therefore, the dilution of the optical signal islargely eliminated, allowing much earlier detection. This effect isdirectly related to the number of fluid partitions formed from theinitial sample/reagent pool. The effect of the number of fluidpartitions on the number of cycles required for detection can bedescribed by the following Equation E1:

$N = \frac{\ln\;{{D_{L}{A_{N}\left( \frac{V}{X} \right)}}}}{\ln(2)}$where: N=number of cycles; D_(L),=detection limit for optical signal[moles/liter]; X=initial number of DNA molecules; V=volume containingDNA molecules [liters]; A_(N)=Avagadro's number [6.023×1023molecules/mole]. From Equation E1 it is clear that N, the number ofcycles until detection, decreases as V, the partitioned fluid volume,decreases.

The system 100 reduces the duration of each temperature cycle byeffectively increasing the concentration of reactants by enclosing themin picoliter type volumes. Since reaction rates depend on theconcentration of the reactants, the efficiency of a partitioned fluidvolume or droplet should be higher than in an ordinary vessel (such as atest tube) where the reactant quantity (DNA quantity) is extremely low.It is estimated that through the reduction in the number of cycles andthe reduction in the time required for each cycles that the FPDDtechnique can reduce the detection time by an order of magnitude ascompared to bulk solution DNA detection techniques.

The system 100 facilitates removal of interference from competing DNAtemplates. Given the extremely small volumes involved withFluid-Partitioned DNA Detection (FPDD), it is possible to isolate asingle template of the target DNA in a given partitioned volume ormicrodroplet. For example, the formation of 2000 partitioned fluidvolumes or microdroplets (each with a volume of 5×10′9 liters) made bydividing a bulk solution of 10 microliters containing 200 DNA molecules,would result in one DNA molecule per microdroplet on average. This makesit possible to amplify only one template in mixtures containing manykinds of templates without interference. This is extremely important inprocessing of real world aerosol samples containing complex mixtures ofDNA from many sources, and has direct application in screening of cDNAlibraries. The system 100 facilitates removal of interference fromcompeting DNA templates. Given the extremely small volumes involved withFluid-Partitioned DNA Detection (FPDD), it is possible to isolate asingle template of the target DNA in a given partitioned volume ormicrodroplet. For example, the formation of 2000 partitioned fluidvolumes or microdroplets (each with a volume of 5×10′9 liters) made bydividing a bulk solution of 10 microliters containing 2000 DNAmolecules, would result in one DNA molecule per microdroplet on average.This makes it possible to amplify only one template in mixturescontaining many kinds of templates without interference. This isextremely important in processing of real world aerosol samplescontaining complex mixtures of DNA from many sources, and has directapplication in screening of cDNA libraries.

Referring now to FIG. 2, a flow diagram of another embodiment of asystem constructed in accordance with the present invention isillustrated. The system is designated generally by the reference numeral200. The flow diagram illustrating system 200 shows block 201“partitioning” the sample, block 202 performing “PCR” on the sample, andblock 203 “detection and analysis.” The system 200 provides a method andapparatus for performing extremely rapid nucleic acid amplification anddetection. The system 200 provides an apparatus for nucleic acidamplification of a sample comprising means for partitioning the sampleinto partitioned sections, means for performing PCR on the partitionedsections, and means for detection and analysis of the partitionedsections. The system 200 also provides a method of nucleic acidamplification of a sample comprising the steps of partitioning thesample into partitioned sections, subjecting the partitioned sections toPCR, and detecting and analyzing the partitioned sections of the sample.

In block 201 a chemical reagent and an input sample are “partitioned”into a large number of microdroplets or other forms of fluid partitionsprior to amplification. The system 200 achieves a reduction in the totalnumber of cycles by limiting the dilution of the optically generatedsignal (e.g., fluorescence or absorption). The formation of partitionedfluid volumes of the DNA-containing solution effectively isolates thefluid volumes which contain the target DNA from the fluid volumes thatdo not contain the target DNA. Therefore, the dilution of the opticalsignal is largely eliminated, allowing much earlier detection. Thiseffect is directly related to the number of fluid partitions formed fromthe initial sample/reagent pool.

In block 202 selected portions of each nucleic acid sample are thenamplified using polymerase chain reaction (PCR), with the productcontained in each partitioned fluid volume. This results in much moreconcentrated amplification product, since the volume containing thereaction is so small. If a Taqman type detection approach is used,fluorescent dye molecules unquenched by the PCF amplification are alsomore concentrated, making possible earlier optical based detection.Since it is possible to contain very amounts of the starting target DNAin each partition fluid volume, inhibitory competition fromnear-neighbor DNA templates is less allowing screening of very dilutesamples.

In block 203 partitioned portions of the sample are detected bymonitoring for the calorimetric indicator (e.g., fluorescence or opticalabsorption) generated with each DNA template duplication sequence. Thepartitioned portions of the sample are optically probed to detect thecolorimetric indicator which signals the presence of the target DNA. Thepartitioned portions of the sample can also be scanned optically todetect the colorimetric indicator signaling the presence of the targetDNA. In one embodiment, fluorescence, generated by degradation of thedye/quencher pair on the primer, is detected using a confocal imagingsystem such as that employed in conventional flow cytometers. Scatteringprofiles from individual microdroplets, as in conventional flowcytometers, can be used to eliminate background signal from otherparticles. In block 203 partitioned portions of the sample are detectedby monitoring for the colorimetric indicator (e.g., fluorescence oroptical absorption) generated with each DNA template duplicationsequence. The partitioned portions of the sample are optically probed todetect the colorimetric indicator which signals the presence of thetarget DNA. The partitioned portions of the sample can also be scannedoptically to detect the colorimetric indicator signaling the presence ofthe target DNA. In one embodiment, fluorescence, generated bydegradation of the dye/quencher pair on the primer, is detected using aconfocal imaging system such as that employed in conventional flowcytometers. Scattering profiles from individual microdroplets, as inconventional flow cytometers, can be used to eliminate background signalfrom other particles.

The system 200 has application wherever current PCR-type systems exist,including medical, drug-discovery, biowarfare detection, and otherrelated fields. Biowarfare detection applications include identifying,detecting, and monitoring bio-threat agents that contain nucleic acidsignatures, such as spores, bacteria, etc. Biomedical applicationsinclude tracking, identifying, and monitoring outbreaks of infectiousdisease. The system 200 provides rapid, high throughput detection ofbiological pathogens (viruses, bacteria, DNA in biological fluids,blood, saliva, etc.) for medical applications. Forensic applicationsinclude rapid, high throughput detection of DNA in biological fluids forforensic purposes. Food and beverage safety applications includeautomated food testing for bacterial contamination.

Referring now to FIG. 3, a diagram of another embodiment of a systemconstructed in accordance with the present invention is illustrated. Thesystem is designated generally by the reference numeral 300. The system300 provides an instrument for performing Fluid-Partitioned DNADetection (FPDD) with PCR based detection and amplification. The system300 includes a partitioning section 301, a PCR section 302, and adetection and analysis section 303.

The partitioning section 301 includes a sample introduction unit 304 anda unit 305 where the sample and a PCR reagent are combined. The sampleand a PCR reagent are injected through a small orifice 306. Theinjection of the sample through the small orifice 306 producesmicrodroplets 308.

The PCR section 302 includes a continuous tube 309 for circulating themicrodroplets 308 and suspended in an immiscible carrier fluid 314. Themicrodroplets 308 suspended in an immiscible carrier fluid 314 arepumped through the continuous tube 309 by pump 311. The microdroplets308 suspended in an immiscible carrier fluid 314 are cycled throughheater 310 and cooler 315 to perform PCR.

The detection and analysis section 303 includes a blue laser 312 and adetector 313. The laser 312 is projected upon the droplets 308 as theypass through tube 308 between the laser 312 and the detector 313.

In the system 300, the DNA-containing solution is partitioned into manymicrodroplets 308 and suspended in an immiscible carrier fluid 314. Themicrodroplets 308 are formed by forcing the PCR mix (sample and reagent)through the small orifice or microjet 306. These microdroplets 308 arethen captured in the immiscible fluid 314, such as mineral oil, andflowed past the heating element 310 and cooler 315. An optical signal(e.g., fluorescence or optical absorption), generated by degradation ofthe dye/quencher pair on the primer, is detected using a confocalimaging system such as that employed in conventional flow cytometers.Scattering profiles from individual microdroplets, as in conventionalflow cytometers, can be used to eliminate background signal from otherparticles. Once exposed to multiple heating cycles, the microdropletscan be identified and probed for an optical signal at rates of severalthousand per second.

The FPDD system achieves a reduction in the total number of cycles bylimiting the dilution of the optically generated signal (e.g.,fluorescence or absorption). The formation of partitioned fluid volumesof the DNA-containing solution effectively isolates the fluid volumeswhich contain the target DNA from the fluid volumes that do not containthe target DNA. Therefore, the dilution of the optical signal is largelyeliminated, allowing much earlier detection. This effect is directlyrelated to the number of fluid partitions formed from the initialsample/reagent pool. The effect of the number of fluid partitions on thenumber of cycles required for detection is described by the Equation E1set out earlier.

The FPDD technique reduces the duration of each temperature cycle byeffectively increasing the concentration of reactants by enclosing themin picoliter type volumes. Since reaction rates depend on theconcentration of the reactants, the efficiency of a partitioned fluidvolume or droplet should be higher than in an ordinary vessel (such as atest tube) where the reactant quantity (DNA quantity) is extremely low.It is estimated that through the reduction in the number of cycles andthe reduction in the time required for each cycles that the FPDDtechnique can reduce the detection time by an order of magnitude ascompared to bulk solution DNA detection techniques

The FPDD technique facilitates removal of interference from competingDNA templates. Given the extremely small volumes involved with FPDD, itis possible to isolate a single template of the target DNA in a givenpartitioned volume or microdroplet. For example, the formation of 2000partitioned fluid volumes or microdroplets (each with a volume of 5×10⁻⁹liters) made by dividing a bulk solution of 10 microliters containing200 DNA molecules, would result in one DNA molecule per microdroplet onaverage. This makes it possible to amplify only one template in mixturescontaining many kinds of templates without interference. This isextremely important in processing of real world aerosol samplescontaining complex mixtures of DNA from many sources, and has directapplication in screening of cDNA libraries. The FPDD techniquefacilitates removal of interference from competing DNA templates. Giventhe extremely small volumes involved with FPDD, it is possible toisolate a single template of the target DNA in a given partitionedvolume or microdroplet. For example, the formation of 2000 partitionedfluid volumes or microdroplets (each with a volume of 5×10⁻⁹ liters)made by dividing a bulk solution of 10 microliters containing containing2000 DNA molecules, would result in one DNA molecule per microdroplet onaverage. This makes it possible to amplify only one template in mixturescontaining many kinds of templates without interference. This isextremely important in processing of real world aerosol samplescontaining complex mixtures of DNA from many sources, and has directapplication in screening of cDNA libraries.

With this new bioassay technique, each partitioned DNA-containing fluidvolume contains the necessary biochemical constituents for selectivelyamplifying a specified portion of a sample DNA via polymerase chainreaction (PCR). The target DNA is detected by monitoring for thecolorimetric indicator (e.g., fluorescence or optical absorption)generated with each DNA template duplication sequence.

The system 300 provides a fast, flexible and inexpensive highthroughput, bioassay technology based on creation and suspension ofmicrodroplets in an immiscible carrier stream. Each microdropletcontains the necessary biochemical constituents for selectivelyamplifying and fluorescently detecting a specified portion of a sampleDNA via polymerase chain reaction (PCR). Once exposed to multipleheating cooling cycles, the microdroplets can be identified and probedfor fluorescent signal at rates of several thousand per second.

Isolating the PCR reaction in such small (picoliter) volumes provides anorder of magnitude reduction in overall detection time by:

-   -   (1) reducing the duration of each temperature cycle—the        concentration of reactants increases by enclosing them in        picoliter type volumes. Since reaction kinetics depend on the        concentration of the reactant, the efficiency of a microdroplet        should be higher than in an ordinary vessel (such a test tube)        where the reactant quantity is infinitesimal    -   (2) reducing the total number of cycles—dilution of the        fluorescently generated signal is largely eliminated in such a        small volume, allowing much earlier detection. This effect is        directly related to the number of microdroplets formed from the        initial sample/reagent pool. Since PCR is an exponential        process, for example, 1000 microdroplets would produce a signal        10 cycles faster than typical processing with bulk solutions.    -   (3) removing interference from competing DNA templates—given the        extremely small volumes involved, it is possible to isolate a        single template of the target DNA in a given microdroplet. A pL        microdoplet filled with a 1 pM solution, for example, will be        occupied by only one molecule on average. This makes it possible        to amplify only one template in mixtures containing many kinds        of templates without interference. This is extremely important        in processing of real world aerosol samples containing complex        mixtures of DNA from many sources, and has direct application in        screening of precious cDNA libraries.

Referring now to FIG. 4, an illustration of another embodiment of asystem constructed in accordance with the present invention isillustrated. The system is designated generally by the reference numeral400. The system 300 provides system for nucleic acid amplification of asample. The system 400 includes means for partitioning the sample intopartitioned sections and means for performing PCR on the partitionedsections of the sample.

The sample is separated into immiscible slugs 406, 407, and 408. Theimmiscible slugs 406, 407, and 408 are formed through a system ofmicrofluidics. Background information on microfluidics is contained inU.S. Pat. No. 5,876,187 for micropumps with fixed valves to Fred K.Forster et al., patented Mar. 2, 1999. As stated in U.S. Pat. No.5,876,187, “Miniature pumps, hereafter referred to as micropumps, can beconstructed using fabrication techniques adapted from those applied tointegrated circuits. Such fabrication techniques are often referred toas micromachining. Micropumps are in great demand for environmental,biomedical, medical, biotechnical, printing, analytical instrumentation,and miniature cooling applications.” Microchannels 403, 404, and 405 areformed in substrates 401 and 402. The disclosures of U.S. Pat. Nos.5,876,187 and 5,876,187 are incorporated herein by reference.

The immiscible slugs 406, 407, and 408 can be moved through themicrochannels using magnetohydrodynamics. Background information onmagnetohydrodynamics is contained in U.S. Pat. No. 6,146,103 formicromachined magnetohydrodynamic actuators and sensors to Abraham P.Lee and Asuncion V. Lemoff, patented Nov. 14, 2000. As stated in U.S.Pat. No. 6,146,103, “Microfluidics is the field for manipulating fluidsamples and reagents in minute quantities, such as in micromachinedchannels, to enable handheld bioinstrumentation and diagnostic toolswith quicker process speeds. The ultimate goal is to integrate pumping,valving, mixing, reaction, and detection on a chip for biotechnological,chemical, environmental, and health care applications. Most micropumpsdeveloped thus far have been complicated, both in fabrication anddesign, and often are difficult to reduce in size, negating manyintegrated fluidic applications. Most pumps have a moving component toindirectly pump the fluid, generating pulsatile flow instead ofcontinuous flow. With moving parts involved, dead volume is often aserious problem, causing cross-contamination in biological sensitiveprocesses. The present invention utilizes MHDs for microfluid propulsionand fluid sensing, the microfabrication methods for such a pump, and theintegration of multiple pumps for a microfluidic system. MHDs is theapplication of Lorentz force law on fluids to propel or pump fluids.Under the Lorentz force law, charged particles moving in a uniformmagnetic field feel a force perpendicular to both the motion and themagnetic field. It has thus been recognized that in the microscale, theMHD forces are substantial for propulsion of fluids throughmicrochannels as actuators, such as a micropump, micromixer, ormicrovalve, or as sensors, such as a microflow meter, or viscositymeter. This advantageous scaling phenomenon also lends itself tomicromachining by integrating microchannels with micro-electrodes.” Thedisclosure of U.S. Pat. No. 6,146,103 is incorporated herein byreference.

The means for performing PCR on the partitioned sections of the samplecan be a system for alternately heating and cooling the immiscible slugs406, 407, and 408. Alternatively, the means for performing PCR on thepartitioned sections of the sample can be a system for alternatelyheating and cooling the immiscible slugs 406, 407, and 408 can be asystem for moving the immiscible slugs 406, 407, and 408 through zonesfor heating and cooling. An example of such a system is shown in U.S.patent application No. 2002/0127152 published Sep. 12, 2002 for aconvectively driven PCR thermal-cycling system described as follows: “Apolymerase chain reaction system provides an upper temperature zone anda lower temperature zone in a fluid sample. Channels set up convectioncells in the fluid sample and move the fluid sample repeatedly throughthe upper and lower temperature zone creating thermal cycling.” Thedisclosure of U.S. Patent Application No. 2002/0127152 is incorporatedherein by reference.

In another embodiment of the invention, the DNA-containing solution ispartitioned by adding a gelling agent to the solution to form cells ofpartitioned volumes of fluid separated by the gelling agent. Using thisapproach for fluid partitioning, the DNA-containing solution is gelledin a tube or as a very thin layer. For example, it can be in a thinlayer between flat plates and the surface of the thin film can beoptically probed spatially in directions parallel to the film surface todetect micro-regions in the film where the colorimetric indicatorsuggests the presence of the target DNA.

Another embodiment of the invention is to partition the DNA-containingsolution as microdroplets in an immiscible fluid where the droplets arearranged in a two-dimensional array such that the array of microdropletscan be optically probed to detect the colorimetric indicator whichsignals the presence of the target DNA. In this approach a solidhydrophobic substrate supports the microdroplets. For example, in smallindentations, and the immiscible “partitioning” fluid is less dense thanthe aqueous DNA-containing solution.

In another embodiment of the invention the DNA-containing solution ispartitioned using mechanical means. For example, the DNA-containingsolution can be partitioned into an array of capillaries, microtubes, orwells. In this approach, the micro vessels holding each partitionedfluid volume can be scanned optically to detect the colorimetricindicator signaling the presence of the target DNA.

Referring now to FIGS. 5A, 5B, and 5C example representations of themechanical partitioning approach for DNA detection using fluidpartitioning are shown. In FIG. 5A a line of capillaries or micro-tubes501 are used for partitioning and holding the DNA containing solution.In FIG. 5B an array 502 of capillaries or micro-tubes are used forpartitioning the DNA-containing solution. In FIG. 5C a microwells ormicro-vessels unit 503 is used for partitioning and holding theDNA-containing solution.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and have been described in detail herein.However, it should be understood that the invention is not intended tobe limited to the particular forms disclosed. Rather, the invention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the followingappended claims.

The invention claimed is:
 1. An apparatus for nucleic acid amplificationof a sample, comprising: means for partitioning said sample intopartitioned sections, wherein said means for partitioning said sampleinto partitioned sections comprises an injection orifice, and means forperforming PCR on said partitioned sections of said sample.
 2. Theapparatus for nucleic acid amplification of a sample of claim 1 whereinsaid injection orifice is an injection orifice that producesmicrodroplets.
 3. The apparatus for nucleic acid amplification of asample of claim 1 wherein said injection orifice is an injection orificethat injects said sample and a PCR reagent.
 4. The apparatus for nucleicacid amplification of a sample of claim 1 wherein said means forperforming PCR on said partitioned sections of said sample comprises acontinuous tube for circulating said partitioned sections of said samplethrough a heater to perform PCR.
 5. The apparatus for nucleic acidamplification of a sample of claim 1 wherein said means for performingPCR on said partitioned sections of said sample comprises a continuoustube for circulating said partitioned sections of said sample through aheater and cooler to perform PCR.
 6. The apparatus for nucleic acidamplification of a sample of claim 1 wherein said means for performingPCR on said partitioned sections of said sample comprises a pump, acontinuous tube, and a heater.
 7. The apparatus for nucleic acidamplification of a sample of claim 1 including means for detection andanalysis of said partitioned sections of said sample comprising a laserand a detector.
 8. The apparatus for nucleic acid amplification of asample of claim 1 including means for detection and analysis of saidpartitioned sections of said sample comprising a blue laser and adetector.
 9. The apparatus for nucleic acid amplification of a sample ofclaim 1 wherein said means for partitioning said sample into partitionedsections comprises means for separating said sample into immiscibleslugs.
 10. A method of nucleic acid amplification of a sample,comprising the steps of: partitioning said sample into partitionedsections, wherein said step of partitioning said sample into partitionedsections comprises flowing said sample through an injection orifice, andsubjecting said partitioned sections of said sample to PCR.
 11. A methodof nucleic acid amplification of a sample, the method comprising thesteps a. providing an aqueous solution, wherein the aqueous solutioncomprises components for performing nucleic acid amplification and aninput sample comprising a plurality of nucleic acids; b. providing animmiscible partitioning fluid; c. providing a solid substrate, whereinthe solid substrate comprises a two-dimensional array of smallindentations; d. contacting the aqueous solution with the immisciblepartitioning fluid on the solid substrate, wherein the immisciblepartitioning fluid is less dense than the aqueous solution, wherein thesolid substrate is configured such that the contacting the aqueoussolution with the immiscible partitioning fluid partitions the inputsample comprising the plurality of nucleic acids into one or moremicrodroplets, and wherein the one or more microdroplets are furtherpartitioned into and held by the two-dimensional array of smallindentations; and e. performing nucleic acid amplification of the one ormore microdroplets held by the two-dimensional array of smallindentations.
 12. The method of claim 11, wherein the solid substrate ishydrophobic.
 13. The method of claim 11, wherein the nucleic acidamplification step comprises alternately heating and cooling the solidsubstrate.
 14. The method of claim 11, wherein the nucleic acidscomprise a target DNA.
 15. The method of claim 12, wherein the one ormore microdroplets contain, on average, a single template of the targetDNA, and wherein the single template is amplified within the one or moremicrodroplets.
 16. The method of claim 11, wherein the components forperforming nucleic acid amplification comprise one or more polymerasechain reaction (PCR) reagents.
 17. The method of claim 11, wherein themethod further comprises detecting one or more products of the nucleicacid amplification.
 18. The method of claim 17, wherein the detectingcomprises optically detecting.
 19. The method of claim 18, wherein theoptically detecting comprises confocal imaging.
 20. The method of claim18, wherein the optically detecting comprises laser excitation.
 21. Themethod of claim 18, wherein the optically detecting comprisesfluorescent detection.
 22. The method of claim 18, wherein the opticallydetecting comprises detecting a colorimetric indicator.
 23. The methodof claim 18, wherein the optically detecting comprises signaling thepresence of a target nucleic acid.
 24. The method of claim 11, whereinthe nucleic acid amplification comprises multiple heating and coolingcycles.
 25. The method of claim 24, wherein the number of cycles issufficient to detect products of the nucleic acid amplification.
 26. Themethod of claim 11, wherein the one or more microdroplets have a volumeof about 5×10⁻⁹ to 10⁻¹² liters.